Intrinsic factor preparation



United States Patent INTRINSIC FACTOR PREPARATION Kenneth C. Robbins, Chicago, Ill., assignor to Armour and Company, Chicago, 111., a corporation of Illinois No Drawing. Application March 23, E55 Serial No. 497,401

4 Claims. (Cl. '167--74) This invention relates to a method of obtaining intrinsic factor preparations of enhanced potency and preparations obtained thereby. The method of this invention has particular utility in producing intrinsic factor concentrates from hog duodenum and hog stomach mucosa, but it can also be advantageously applied to other intrinsic factor-bearing mammalian tissues.

This is a continuation in part of my co-pending patent application Serial No. 310,372, filed September 18, 1952, now Pat. No. 2,770,570.

It is known that at least duodenum and stomach tissues of hogs contain a substance which is of value in the treatment of anemia in humans, and particularly in the treatment of pernicious anemia. The value of this substance can be most dramatically demonstrated for pernicious anemia patients in relapse, and the efficacy of a particular hog duodenum or stomach preparation in treating such patients has been accepted as the most reliable method for assaying the potency of the preparation.

The substance referred to above is generally designated as the intrinsic factor or as Castles intrinsic factor. However, the intrinsic factor has not been isolated, and therefore its exact nature is still unknown. For the purpose of this application the term intrinsic factor will be used to designate the component (or components) in hog duodenal or stomach tissues which is of value in the treatment of anemic conditions, particularly of the macrocytic type.

Heretofore various intrinsic factor preparations have been utilized in the treatment of pernicious anemia, but all of them have suffered from the defects of requiring the patient to swallow objectionably large quantities of unpleasant material. For example, the average daily re quirement of whole hog duodenum by a pernicious anemia patient is about one-fourth to one-half pound, and even when desiccated at least 20 to 40 grams are required. Efforts have been made to improve this situation by attempting to prepare more concentrated intrinsic factor preparations, and this has resulted in prep arations which are adequate in daily quantities of as low as 2 grams. However, when the previously known concentrates are employed in less than 1 gram per day closes on pernicious anemia patients in relapse, a full response is notobtained. Daily doses of the order of 1 to 2 grams are objectionable from the standpoint of the patient. For example, to administer 1 gram of material, about 5 tablets or capsules are required. The recognized objective has been to prepare a concentrate which would give a full response by the intake of one small tablet per day, that is, of the order of 200 milligrams or less, but until now this has not been achieved in spite of considerable efforts towards solving the problem.

It is therefore a general object of this invention to prepare intrinsic factor preparations of enhanced poice Preparatory to extracting the intrinsic factor from in-.

trinsic-bearing tissue, it is preferred to comminute the tissue by hashing, grinding, etc. Also, the fresh intrinsic factor-bearing tissue can be frozen and then ground in the presence of Dry Ice to prevent any possibility of loss in intrinsic factor activity. in utilizing the mucosa of stomach and duodenum, the mucus lining thereof can be excised and comminuted by the aforementioned methods preparatory to extraction. The pyloric mucosa forms an inner lining at the posterior end of the stomach proximate to the pyloric valve, and may be differentiated from the cardiac and fundus portions of such stomach lining by its deep red color. The pyloric mucosa starting material for this process maybe obtained by separating at least another portion of the stomach mucosa from the pyloric portion by excision (as by the removal of the fundus or cardiac portion of the mucosa from the pyloric portion of the mucosa), and subjecting such pyloric portion to comminution prior to.intrinsic factor extraction.

The comminuted intrinsic factor-bearing tissue may be extracted with water to obtain an aqueous extract containing the intrinsic factor. Thevolume of water employed is not particularly critical, but a convenient range is from 2 to 4 volumes of water to each volume of intrinsic factor-bearing tissue. It is preferred to carry out the aqueous extraction at an acidic pH not substantially below .pH 2. To minimize the formation of a colloidal tissue slurry, this extraction may be carried out at a pH not over 5, Le, from pH 2 to S, and optimum results can be obtained ata pH of about 3 to 4. The extraction may be performed by any of the usual procedures, such as agitating the suspensionof tissue in water with a propeller-type stirrer, pump, etc. Inorder to prevent loss of activity it is believed desirable to keep the temperature below 15 (3., although somewhat higher temperatures can probably be employed. Excellent results are obtained at temperatures below 5 C.

The aqueous extract of the intrinsic factor (or super natant) is separately from the tissue residue by any suitable method such as filtration, centrifugation, etc. The aqueous extract is next fractionated with ethanol to obtain as aprecipitate an intrinsic factor concentrate. This step is believed to be largely responsible for the greatly improved results of the process of this invention. Instead of ethanol other liquid organic precipitating agents can be employed such as. the aliphatic monohydric alcohols having less than five carbon atoms and acetone. Other lower aliphatic ketones, such as methyl ethyl ketone, i can be substituted for acetone. Methanol is an especially desirable substitute for ethanol.

This fractionation step may be carried out at an organic precipitating solvent concentration of from 20 .to 60% by volume. Better results are achieved when the precipitating solvent concentration in this step is more than 30% by volume and optimum results are obtained at concentration of such solvent between 45 and by volume. I have found that at a pH of below 4.0 and above 6.0, at an ethanol concentration of 50% and an ionic strength of 0.075, the intrinsic factor substance cannot be precipitated, and consequently the pH in this fractionation step should be from pH 4 to'6. However,

3 I have found that better fractionation of the intrinsic factor is achieved at a pH of from 4.5 to 5.5, and optimum results are achieved at a pH of about 5.0. When the intrinsic factor is derived from pyloric mucosa, I have found that especially desirable alcohol fractionation is obtained at a pH of from 5.0 to 5.4. Also, I have found that at an ionic strength of 0.2 the intrinsic factor cannot be precipitated in the solvent mixture, and consequently the ionic strength therein should be less than 0.15 to precipitate substantially all of the intrinsic factor. However. fractionation of the intrinsic factor may be obtained with fair results above this ionic strength range. The presence of a relatively high concentration of organic precipitating solvent in this fractionation mixture indicates that the temperature of the solvent mixture. should be maintained below C. Better results are achieved when the temperature of this solvent mixture is below 3 C., and especially desirable fractionation of the intrinsic factor is obtained at a temperature of about 5 C., although at a solvent concentration of more than 50% it may be desirable to use a somewhat lower temperature in order to prevent denaturation of the intrinsic factor.

This fractionation step may be carried out by adding to the intrinsic factor extract the organic precipitating solvent, such as ethanol, in such volume as to produce the critical solvent concentration in the resulting mixture. Also, the temperature of the organic precipitating solvent added to the aqueous intrinsic factor extract may be such as to produce the desired temperature in the solvent mixture. For example, the temperature of the organic solvent may be about 20 C. to produce a temperature in the solvent mixture of about -5 C. The result of the above fractionation step is that a precipitate forms which consistsof an intrinsic factor preparation of enhanced potency, and in fact, of higher potency than any preparation which has heretofore been known. The intrinsic factor containing precipitate is separated from the supernatant (filtration, centrifugation, etc.) and is prepared for j medicinal use. For example, the separated precipitate can be removed by centrifugation, and is dissolved in 3 volumes of ice cold Water. The solution is clarified and lyophilized.

The pH adjustment in the extraction step can be made with hydrochloric acid or other suitable acids, such as sulphuric, phosphoric, lactic, citric, etc. The pH adjustment in the fractionation step can be made with sodium hydroxide or other alkali metal hydroxides or an alkaline earth metal hydroxide or carbonate such as potassium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, etc.

One procedure for carrying out this fractionation step involves first adjusting the pH of the aqueous intrinsic factor extract, which is obtained from intrinsic factorbearing tissue, to a pH between 5 and 5.4 prior to the addition thereto of the organic precipitating solvent. This pH adjustment may result in the formation of a small amount of precipitate in the extract, which precipitate is very low in intrinsic factor content. Therefore, it is preferred to remove this precipitate prior to the addi tion thereto of the organic precipitating solvent by centrifugation, filtration, etc. Following the separation of this low potency precipitate from the intrinsic factor extract, the organic solvent may be combined with such extract to a final concentration of from to by volume thereby obtaining as a precipitate the intrinsic factor in concentrated form.

This invention may be more fully illustrated by the following detailed examples:

Example I The following steps are recommended for isolating the intrinsic factor of Castle from hog duodenum:

(1) Suspend 1 volume of fresh frozen comminuted hog duodenum in 3 volumes of ice cold distilled water.

(2) Adjust the pH of the suspension to 3.5121 with 1 N HCl.

(3) Agitate the suspension for 2 hours at about 1 C.

(4) Remove the residue from the extract by centrifugation at about 1 C. and discard it.

(5) Adjust the pH of the supernatant to 4.7 :1 with l N NaOl-I, and separate the resulting low potency precipitate by filtration.

(6) Bring the solution to 40% ethanol concentration at "5 C. with ethanol and hold overnight at 5" C.

(7) Remove the precipitate by concentration.

(8) Dissolve the precipitate in 3 volumes of ice cold water, clarify, and lyophilize.

The final product obtained by the above procedure will be an intrinsic factor preparation of enhanced potency.

Example [I The following constitutes an actual experimental example. 77.25 kg. of hog duodenal tissue were suspended in 230 liters of distilled water at a pH of 3.5 (HCl) and an extraction was carried out at 1 C. for two hours. The extract was separated from the residue by centrifugation at 1 C. The extract having a volume of 200 liters was then adjusted to pH 4.7 (NaOi-I) and held overnight at 1 C. The precipitate thus formed was separated from the supernatant'by filtration at 1 C. and discarded. The supernatant (140 liters) was brought to 40% ethanol by adding 95% ethanol thereto and held overnight at -5 C. The precipitate was separated by centrifugation at -5 C., and the supernatant was discarded. The precipitate thus obtained weighed 920 grams. This precipitate was further processed by dissolving it in 4 liters of ice cold water. The solution was clarified and lyophilized. The lyophilized preparation having a weight of 142 grams was found to be a high potency concentrate of the intrinsic factor of Castle by administering portions thereof to pernicious anemia patients in relapse.

In one series of tests with the product obtained above, the intrinsic factor concentrate was formulated into tablets and capsules in admixture with vitamin B One pernicious anemia patient in relapse reveived 600 milligrams of the intrinsic factor concentrate prepared above plus 8 micrograms of B per day, and evidenced a full U.S.P. response. Another patient showed a full response on a daily intake of 300 milligrams of the intrinsic factor concentrate together with 8 micrograms of B Still another patient showed a full response on a daily intake of milligrams of the intrinsic factor concentrate plus 30 micrograms of B In other words, the above tests indicate that a full USP. response could be expected from pernicious anemia patients in relapse by giving them one small tablet or capsule per day containing as little as 120 milligrams of the intrinsic factor concentrate prepared by the procedure set out in this example, together with 30 micrograms of vitamin B Example 111 An intrinsic factor concentrate of extremely high potency was prepared from porcine pyloric mucosa by the following method. The pyloric mucosa was prepared by washing slime from the inner lining of hog' stomachs with warm water. Then, the pyloric portion of the mucosa, i.e. deep red color portion of the washed hog stomach was separated from the other portions thereof by a cutting operation. The excised pyloric mucosa was frozenwith Dry Ice and comminuted. One kg. of this comm'muted pyloric tissue was mixed with 2 1 of ice-cold water, and the pH of the resulting slurry was adjusted to 3.5 by the addition thereto of 8 N hydrochloric acid. This extraction slurry was then stirred overnight at a temperature of about 4 C. Thereafter, the

bulk of the tissue residue was separated from the supernatant liquid by filtration through cheese-cloth. This supernatant liquid was clarified by centrifugation, and the separated precipitate discarded.

A portion of this clarified extract was dried by lyophilization, and the lyophilized product tested clinically for intrinsic factor activity. This product was found to be clinically active at a daily dose of 75 mg., in combination with 15 mcg. of vitamin B according to the standard U.S.P. assay for intrinsic factor. The yield of dry product was 30 gms., i.e., 30 gms. per kg. of pyloric mucosa tissue. On the basis of the clinical activity of the product, it was calculated that 400 clinical doses of intrinsic factor could be obtained per kg. of pyloricmucosa at the 15 meg. vitamin B level of therapeusis.

The clarified intrinsic factor extract was concentrated by distillation in vacuo to a total solids concentration therein of 8 to 10%. The pH of this aqueous concentrate was adjusted to pH 5.2 by adding thereto a sodium acetate-acetic acid buifer. Thus the aqueous concentrate had a final total solids content of 6% at an ionic strength of 0.15 and a temperature of 4 C. 3 A 95% ethanol was cooled to a temperature of 20 C., and then added to the intrinsic factor concentrate to a final volume of 50%. The temperature of this ethanol mixture was -5 C., while the ionic strength thereof was 0.075 and the total solids content 3%. This ethanol mixture was held overnight at a temperature of 5 C. Thereafter, the precipitate formed in such mixture was separated from the supernatant liquid by centrifugation at a speed of 4,000 rpm. for a period of 30 minutes and a temperature of 5 C. The supernatant liquid was discarded, and the separated precipitate was mixed with about 4 volumes of ice-cold water at a temperature of 4 C. The resulting suspension was mixed by stirring for several hours, and then lyophilized. The yield of dry product was 5 gms, i.e. 5 gms. per kg. of pyloric mucosa. This preparation was tested clinically and found to be active at a daily dosage level of 35 mgs. in combination with mcg. of vitamin B On the basis of this clinical analysis, it was computed that about 140 doses of intrinsic factor, at a 15 meg. vitamin B level, could be obtained per kg. of pyloric mucosa.

Example I V The following tabular results demonstrate the efiicient adaptation of this process to the large scale manufacture of intrinsic factor from pyloric mucosa. The process employed in each of these batches was similar to that described in Example III, and the important quantitations are depicted at significant stages in each batch.

Batches Weight of pyloric mucosa (kg) 280 275 Volume of extract (liters) 110 94.9 Solids (percent) 7.4 8.66 Total weight of solids (kg.) 8. 14 8. 22 Protein (percent) 4. 61 4. 56 Total weight of protein (kg.) 5. 07 4.33 Dry weight of alcohol precipitate (gms. per kg. of

pyloric mucosa) 7. 35 (1.8 Solids (percent) 25 21 Protein (percent 40 39 Volume of centritugate (liters) 247 1 216 s ercent 1.33 1.33 Total weight of solids (kg.). 3. 29 2.87 Protein (percent) 1.38 1.62 Total weight of protein tkg.) 3. 4 3. 48 No. of clinical doses per kg. of pyloric tissue (the product of each batch being active at a level of 35 mg. plus 15 ug. of vitamin B1, per day) 210 195 1 Total solids determined as the weight of a lyophilized aliquot.

2 The yield herein was not representative due to an estimated 10% loss in processing, and consequently the equivalent weight of pyloric mucosa actually involved was calculated as 250 kg.

While in the foregoing specification this invention has been set forth in considerable detail for the purpose of illustration, it will be apparent to those skilled in the art that many of these details may be varied widely without departing from the basic concept and spirit of the invention.

I claim:

1. In a process for preparing an intrinsic factor preparation of enhanced potency, the steps of separating the pyloric portion of hog stomach mucosa from the other portions thereof, extracting said pyloric portion with water at a pH of from 3 to 4 to obtain an intrinsic factor ex tract, adjusting the pH of said extract to from pH 5.0 to 5.4, and then fractionating said extract by adding thereto an alcohol of the group consisting of ethanol and methanol until the alcohol concentration of said extract is more than 30% by volume, to obtain an intrinsic factor precipitate of enhanced potency, and separating said precipitate from the supernatant liquid.

2. In a process for preparing an intrinsic factor preparation of enhanced potency, the steps of separating the pyloric portion of hog stomach mucosa from the other portions thereof, extracting said pyloric portion with water at a pH of from 3 to 4 to obtain an intrinsic factor extract, adjusting the pH of said extract to from pH 5.0 to 5.4, and then fractionating said extract by adding thereto an alcohol of the group consisting of methanol and ethanol until the alcohol concentration of said extract is between 45 and 55% by volume, to obtain an intrinsic factor precipitate of enhanced potency, and separating said precipitate from the supernatant liquid.

3. In a process for preparing an intrinsic factor preparation of enhanced potency, the steps of separating pyloric portion of hog stomach mucosa from the other portions thereof, extracting said pyloric portion with water at a pH of from 2 to 5 to obtain an intrinsic factor extract, adjusting the pH of said extract to between pH 5.0 and 5.4, and then fractionating said extract at an ionic strength of not more than 0.15 by adding thereto an alcohol of the group consisting ofethanol and methanol until the alcohol concentration of said extract is from 45 to 55% by volume, to obtain an intrinsic factor precipitate of enhanced potency, and separating said precipitate from the supernatant liquid.

4. In a process for the preparation of an intrinsic factor of enhanced potency, the steps of separating the pyloric portion of hog stomach mucosa from the other portions thereof, extracting said pyloric portion with water at a pH of from 3 to 4 to obtain an intrinsic factor extract, adjusting the pH of said extractive from 4 to 6, and fractionating said extract by adding thereto an alcohol of the group consisting of methanol and ethanol until the alcohol concentration of said extract is between 20 and 60% by volume to obtain an intrinsic factor precipitate of enhanced potency, and separating said precipitate from the supernatant liquid.

References Cited in the file of this patent UNITED STATES PATENTS Fogelson Oct. 27, 1931 Robbins Nov. 13, 1956 OTHER REFERENCES 

1. IN A PROCESS FOR PREPARING AN INTRINSIC FACTOR PREPARATION OF ENHANCED POTENCY, THE STEPS OF SEPARATING THE PYLORIC PORTION OF HOG STOMACH MUCOSA FROM THE OTHER PORTIONS THEREOF, EXTRACTING SAID PYLORIC PORTION WITH WATER AT A PH OF FROM 3 TO 4 TO OBTAIN AN INTRINSIC FACTOR EXTRACT, ADJUSTING THE PH OF SAID EXTRACT TO FROM PH 5.0 TO 5.4 AND THEN FRACTIONATING SAID EXTRACT BY ADDING THERETO NOT UNTIL THE ALCOHOL CONCENTRATION OF SAID EXTRACT IS MORE NOT UNTIL THE ALCOHOL CONCENTRATION OF SAID EXTRACT IS MORE THAN 30% BY VOLUME, TO OBTAIN AN INTRINSIC FACTOR PRECIPITATE OF ENHANCED POTENCY, AND SEPARATING SAID PRECIPITATE FROM THE SUPERNATANT LIQUID. 